Relativistic quantum revivals.

Quantum revivals are now a well-known phenomena within nonrelativistic quantum theory. In this Letter we display the effects of relativity on revivals and quantum carpets. It is generally believed that revivals do not occur within a relativistic regime. Here we show that while this is generally true, it is possible, in principle, to set up wave packets with specific mathematical properties that do exhibit exact revivals within a fully relativistic theory.

Modelling the interdependence between the stoichiometry of receptor oligomerization and ligand binding for a coexisting dimer/tetramer receptor system.

Many G protein-coupled receptors have been shown to exist as oligomers, but the oligomerization state and the effects of this on receptor function are unclear. For some G protein-coupled receptors, in ligand binding assays, different radioligands provide different maximal binding capacities. Here we have developed mathematical models for co-expressed dimeric and tetrameric species of receptors. We have considered models where the dimers and tetramers are in equilibrium and where they do not interconvert and we have also considered the potential influence of the ligands on the degree of oligomerization. By analogy with agonist efficacy, we have considered ligands that promote, inhibit or have no effect on oligomerization. Cell surface receptor expression and the intrinsic capacity of receptors to oligomerize are quantitative parameters of the equations. The models can account for differences in the maximal binding capacities of radioligands in different preparations of receptors and provide a conceptual framework for simulation and data fitting in complex oligomeric receptor situations.

Agonist binding, agonist affinity and agonist efficacy at G protein-coupled receptors.

Measurements of affinity and efficacy are fundamental for work on agonists both in drug discovery and in basic studies on receptors. In this review I wish to consider methods for measuring affinity and efficacy at G protein coupled receptors (GPCRs). Agonist affinity may be estimated in terms of the dissociation constant for agonist binding to a receptor using ligand binding or functional assays. It has, however, been suggested that measurements of affinity are always contaminated by efficacy so that it is impossible to separate the two parameters. Here I show that for many GPCRs, if receptor/G protein coupling is suppressed, experimental measurements of agonist affinity using ligand binding (K(obs)) provide quite accurate measures of the agonist microscopic dissociation constant (KA). Also in pharmacological functional studies, good estimates of agonist dissociation constants are possible. Efficacy can be quantitated in several ways based on functional data (maximal effect of the agonist (E(max)), ratio of agonist dissociation constant to concentration of agonist giving half maximal effect in functional assay (K(obs)/EC50), a combined parameter E(max)K(obs)/EC50). Here I show that E(max)K(obs)/EC50 provides the best assessment of efficacy for a range of agonists across the full range of efficacy for full to partial agonists. Considerable evidence now suggests that ligand efficacy may be dependent on the pathway used to assess it. The efficacy of a ligand may, therefore, be multidimensional. It is still, however, necessary to have accurate measures of efficacy in different pathways.

Analysis of second messenger pathways stimulated by different chemokines acting at the chemokine receptor CCR5.

The chemokine receptor, CCR5, responds to several chemokines leading to changes in activity in several signalling pathways. Here, we investigated the ability of different chemokines to provide differential activation of pathways. The effects of five CC chemokines acting at CCR5 were investigated for their ability to inhibit forskolin-stimulated 3'-5'-cyclic adenosine monophosphate (cAMP) accumulation and to stimulate Ca(2+) mobilisation in Chinese hamster ovary (CHO) cells expressing CCR5. Macrophage inflammatory protein 1alpha (D26A) (MIP-1alpha (D26A), CCL3 (D26A)), regulated on activation, normal T-cell expressed and secreted (RANTES, CCL5), MIP-1beta (CCL4) and monocyte chemoattractant protein 2 (MCP-2, CCL8) were able to inhibit forskolin-stimulated cAMP accumulation, whilst MCP-4 (CCL13) could not elicit a response. CCL3 (D26A), CCL4, CCL5, CCL8 and CCL13 were able to stimulate Ca(2+) mobilisation through CCR5, although CCL3 (D26A) and CCL5 exhibited biphasic concentration-response curves. The Ca(2+) responses induced by CCL4, CCL5, CCL8 and CCL13 were abolished by pertussis toxin, whereas the response to CCL3 (D26A) was only partially inhibited by pertussis toxin, indicating G(i/o)-independent signalling induced by this chemokine. Although the rank order of potency of chemokines was similar between the two assays, certain chemokines displayed different pharmacological profiles in cAMP inhibition and Ca(2+) mobilisation assays. For instance, whilst CCL13 could not inhibit forskolin-stimulated cAMP accumulation, this chemokine was able to induce Ca(2+) mobilisation via CCR5. It is concluded that different chemokines acting at CCR5 can induce different pharmacological responses, which may account for the broad spectrum of chemokines that can act at CCR5.

Mechanisms underlying agonist efficacy.

Agonist efficacy is a measure of how well an agonist can stimulate a response system linked to a receptor. Efficacy can be assessed in functional assays and various parameters (E(max), K(A)/EC(50), E(max).K(A)/EC(50)) determined. The E(max).K(A)/EC(50) parameter provides a good estimate of efficacy across the full range of efficacy. A convenient assay for the efficacy of agonists for some receptors is provided by the [(35)S]GTP[S] (guanosine 5'-[gamma-[(35)S]thio]triphosphate)-binding assay. In this assay, the normal GTP-binding event in GPCR (G-protein-coupled receptor) activation is replaced by the binding of the non-hydrolysable analogue [(35)S]GTP[S]. This assay may be used to profile ligands for their efficacy, and an example here is the D(2) dopamine receptor where an efficacy scale has been set up using this assay. The mechanisms underlying the assay have been probed. The time course of [(35)S]GTP[S] binding follows a pseudo-first-order reaction with [(35)S]GTP[S] binding reaching equilibrium after approx. 3 h. The [(35)S]GTP[S]-binding event is the rate-determining step in the assay. Agonists regulate the maximal level of [(35)S]GTP[S] bound, rather than the rate constant for binding. The [(35)S]GTP[S]-binding assay therefore determines agonist efficacy on the basis of the amount of [(35)S]GTP[S] bound rather than the rate of binding.

Mechanisms of G protein activation via the D2 dopamine receptor: evidence for persistent receptor/G protein interaction after agonist stimulation.

BACKGROUND AND PURPOSE: The aim of this report is to study mechanisms of G protein activation by agonists. EXPERIMENTAL APPROACH: The association and dissociation of guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding at G proteins in membranes of CHO cells stably transfected with the human dopamine D(2short) receptor was studied in the presence of a range of agonists. KEY RESULTS: Binding of [(35)S]GTPgammaS was dissociable in the absence of agonist and dissociation was accelerated both in rate and extent by dopamine, an effect which was blocked by the dopamine D(2) receptor antagonist raclopride and by suramin, which inhibits receptor/G protein interaction. A range of agonists of varying efficacy increased the rate of dissociation of [(35)S]GTPgammaS binding, with the more efficacious agonists resulting in faster dissociation. Agonists were able to dissociate about 70% of the pre-bound [(35)S]GTPgammaS, leaving a component which may not be accessible to the agonist-bound receptor. The dissociable component of the [(35)S]GTPgammaS binding was reduced with longer association times and increased [(35)S]GTPgammaS concentrations. CONCLUSIONS AND IMPLICATIONS: These data are consistent with [(35)S]GTPgammaS binding being initially to receptor-linked G proteins and then to G proteins which have separated from the agonist bound receptor. Under the conditions used typically for [(35)S]GTPgammaS binding assays, therefore, much of the agonist-receptor complex remains in proximity to G proteins after they have been activated by agonist.

Dipolar excitations at the LIII x-ray absorption edges of the heavy rare-earth metals.

We report measured dipolar asymmetry ratios at the LIII edges of the heavy rare-earth metals. The results are compared with a first-principles calculation and excellent agreement is found. A simple model of the scattering is developed, enabling us to reinterpret the resonant x-ray scattering in these materials and to identify the peaks in the asymmetry ratios with features in the spin and orbital moment densities.

Assays for enhanced activity of low efficacy partial agonists at the D(2) dopamine receptor.

BACKGROUND AND PURPOSE: Low efficacy partial agonists at the D2 dopamine receptor may be useful for treating schizophrenia. In this report we describe a method for assessing the efficacy of these compounds based on stimulation of [35S]GTPgammaS binding. EXPERIMENTAL APPROACH: Agonist efficacy was assessed from [(35)S]GTPgammaS binding to membranes of CHO cells expressing D2 dopamine receptors in buffers with and without Na+. Effects of Na+ on receptor/G protein coupling were assessed using agonist/[3H]spiperone competition binding assays. KEY RESULTS: When [35S]GTPgammaS binding assays were performed in buffers containing Na+, some agonists (aripiprazole, AJ-76, UH-232) exhibited very low efficacy whereas other agonists exhibited measurable efficacy. When Na+ was substituted by N-methyl D-glucamine, the efficacy of all agonists increased (relative to that of dopamine) but particularly for aripiprazole, aplindore, AJ-76, (-)-3-PPP and UH-232. In ligand binding assays, substitution of Na+ by N-methyl D-glucamine increased receptor/G protein coupling for some agonists -. aplindore, dopamine and (-)-3-PPP - but for aripiprazole, AJ-76 and UH-232 there was little effect on receptor/G protein coupling. CONCLUSIONS AND IMPLICATIONS: Substitution of Na+ by NMDG increases sensitivity in [(35)S]GTPgammaS binding assays so that very low efficacy agonists were detected clearly. For some agonists the effect seems to be mediated via enhanced receptor/G protein coupling whereas for others the effect is mediated at another point in the G protein activation cycle. AJ-76, aripiprazole and UH-232 seem particularly sensitive to this change in assay conditions. This work provides a new method to discover these very low efficacy agonists.

Intracellular cytokine production by fetal and adult monocytes.

BACKGROUND/PURPOSE: In light of the neonate's increased susceptibility to systemic infection, the authors hypothesized that adult and fetal monocytes have different cytokine expression profiles in response to lipopolysaccharide (LPS), and interleukin (IL)-11, a counter-inflammatory cytokine. METHODS: Samples of cord blood (n = 30) and adult blood (n = 30) were obtained and treated as follows: control (baseline expression), LPS exposure, and IL-11 or IL-11+LPS exposure. After incubation with a protein transport inhibitor, mononuclear cells were stained for intracellular tumor necrosis factor (TNF)-alpha, IL-1beta, IL-6, and IL-8. Each sample was then analyzed by flow cytometry for cytokine expression. Cytokine production was measured by the percent positive as well as the fluorescence index for each cytokine. Analysis of variance (ANOVA) and Students t tests were used for statistical analysis. RESULTS: Baseline levels of IL-8 were significantly higher for fetal monocytes (P <.0001). After LPS exposure, fetal monocytes produced less TNF-alpha (P =.0105) and more IL-8 (P <.0007) relative to adult cells. IL-11 treatment reduced baseline production of IL-8 in fetal and adult monocytes (P <.05). CONCLUSIONS: These results suggest that neonatal monocytes portray a different cytokine expression profile compared with adult monocytes. IL-11 treatment appears to alter the IL-8 expression of resting fetal and adult monocytes.

Agonist regulation of D(2) dopamine receptor/G protein interaction. Evidence for agonist selection of G protein subtype.

The D(2) dopamine receptor has been expressed in Sf21 insect cells together with the G proteins G(o) and G(i2), using the baculovirus system. Expression levels of receptor and G protein (alpha, beta, and gamma subunits) in the two preparations were similar as shown by binding of [(3)H]spiperone and quantitative Western blot, respectively. For several agonists, binding data were fitted best by a two-binding site model in either preparation, showing interaction of expressed receptor and G protein. For some agonists, binding to the higher affinity site was of higher affinity in D(2)/G(o) than in the D(2)/G(i2) preparation. Some agonists exhibited binding data that were best fitted by a two-binding site model in D(2)/G(o) and a one-binding site model in D(2)/G(i2). Therefore, receptor/G protein interaction seemed to be stronger in the D(2)/G(o) preparation. Agonist stimulation of [(35)S]GTP gamma S (guanosine 5'-3-O-(thio)triphosphate) binding in the two preparations also gave evidence for higher affinity D(2)/G(o) interaction. In the D(2)/G(o) preparation, agonist stimulation of [(35)S]GTP gamma S binding occurred at higher potency for several agonists, and a higher stimulation (relative to dopamine) was achieved in D(2)/G(o) compared with D(2)/G(i2). Some agonists were able to stimulate [(35)S]GTP gamma S binding in the D(2)/G(o) preparation but not in D(2)/G(i2). The extent of D(2) receptor selectivity for G(o) over G(i2) is therefore dependent on the agonist used, and thus agonists may stabilize different conformations of the receptor with different abilities to couple to and activate G proteins.

Pharmacodynamic and pharmacokinetic properties of a premixed 85/15 human insulin preparation.

BACKGROUND: Many patients with diabetes use mixtures of fast-acting (regular human) insulin and intermediate-acting (neutral protamine Hagedorn [NPH]) insulin to control their blood glucose levels. Premixed insulin is available in a 70%/30% mixture and a 50%/50% mixture of NPH/regular human insulin. For some patients, however, a premixed formulation containing > or =30% regular human insulin can provide too much fast-acting insulin, potentially causing an increased risk for hypoglycemia in the early hours after injection. OBJECTIVE: The pharmacokinetic and pharmacodynamic properties of a premixed formulation of 85% NPH insulin and 15% regular human insulin (85/15) were compared with those of a premixed 70%/30% NPH/regular human insulin preparation and 100% NPH insulin. METHODS: A 12-hour euglycemic clamp approach was used to assess glucose-lowering effects and serum insulin levels in 36 healthy male volunteers in a single-dose (0.5 U/kg), randomized, double-blind, 3-period, crossover study. RESULTS: From 0 to 8 hours after injection, the glucose-lowering effects and serum insulin levels for the 85/15 premixed insulin preparation were significantly greater than those for NPH insulin (P < or = 0.05) but significantly less than those for the 70/30 premixed insulin preparation. The mean (+/- SEM) maximum glucose infusion rate (GIRmax) was 8+/-0.6 mg/(min x kg) for the 85/15 preparation, 7+/-0.6 mg/(min x kg) for NPH, and 9+/-0.6 mg/(min x kg) for the 70/30 preparation, with time to peak GIR (tmax(GIR)) occurring at 313, 360, and 272 minutes, respectively. Time to peak insulin levels did not differ significantly for the 3 preparations, but maximum serum insulin concentration (Cmax(ins)) was significantly different between the groups (70/30 premix: 54+/-2.2 microU/mL; 85/15 premix: 44+/-2.4 microU/mL; NPH: 35+/-1.7 microU/mL). Glucodynamic effect and serum insulin levels did not differ significantly among preparations during the interval from 8 to 12 hours after injection. Mean serum C-peptide levels ranged from -0.6 to 1.0 ng/mL for each preparation during the 12-hour period after injection. CONCLUSIONS: The 85/15 premixed insulin preparation demonstrated clinical pharmacokinetic and pharmacodynamic properties that were intermediate between, and significantly different from, those of NPH insulin and the 70/30 premixed insulin preparation.

Mechanisms of inverse agonism of antipsychotic drugs at the D(2) dopamine receptor: use of a mutant D(2) dopamine receptor that adopts the activated conformation.

The antipsychotic drugs have been shown to be inverse agonists at the D(2) dopamine receptor. We have examined the mechanism of this inverse agonism by making mutations in residue T343 in the base of the sixth transmembrane spanning region of the receptor. T343R, T343S and T343K mutant D(2) dopamine receptors were made and the T343R mutant characterized in detail. The T343R mutant D(2) dopamine receptor exhibits properties of a receptor that resides more in the activated state, namely increased agonist binding affinity (independent of G-protein coupling and dependent on agonist efficacy), increased agonist potency in functional tests (adenylyl cyclase inhibition) and increased inverse agonist effects. The binding of agonists to the mutant receptor also shows sensitivity to sodium ions, unlike the native receptor, so that isomerization of the receptor to its inactive state may be driven by sodium ions. The binding of inverse agonists to the receptor is, however, unaffected by the mutation. We conclude that inverse agonism at this receptor is not achieved by the inverse agonist binding preferentially to the non-activated state of the receptor over the activated state. Rather the inverse agonist appears to bind to all forms of the receptor but then renders the receptor inactive.

Dopamine D2 receptor dimer formation: evidence from ligand binding.

We have examined the binding of two radioligands ([(3)H]spiperone and [(3)H]raclopride) to D(2) dopamine receptors expressed in Chinese hamster ovary cells. In saturation binding experiments in the presence of sodium ions, both radioligands labeled a similar number of sites, whereas in the absence of sodium ions [(3)H]raclopride labeled about half the number of sites labeled by [(3)H]spiperone. In competition experiments in the absence of sodium ions, however, raclopride was able to inhibit [(3)H]spiperone binding fully. In saturation analyses with [(3)H]spiperone in the absence of sodium ions raclopride exerted noncompetitive effects, decreasing the number of sites labeled by the radioligand. These data are interpreted in terms of a model where the receptor exists as a dimer, and in the absence of sodium ions, raclopride exerts negative cooperativity across the dimer both for its own binding and the binding of spiperone. A model of the receptor has been produced that provides a good description of the experimental phenomena described here.

Efficacy, safety, and pump compatibility of insulin aspart used in continuous subcutaneous insulin infusion therapy in patients with type 1 diabetes.

OBJECTIVE: The purpose of this study was to compare the efficacy, safety and pump compatibility of insulin aspart (a rapid-acting insulin analog) and buffered regular human insulin in patients with type 1 diabetes undergoing continuous subcutaneous insulin infusion (CSII) therapy. RESEARCH DESIGN AND METHODS: This was a single-center randomized open-label study Patients received CSII therapy with insulin aspart (n = 19) or buffered regular human insulin (n = 10) for 7 weeks. Bolus doses of insulin aspart were administered immediately before meals and buffered regular human insulin 30 min before meals. RESULTS: Insulin aspart and buffered regular human insulin were both effective in controlling average daily blood glucose levels (8.2 +/- 1.9 and 8.5 +/- 2.1 mmol/l, respectively) (mean +/- SD) and maintaining serum fructosamine (343 +/- 25.7 and 336 +/- 27.4 micromol/l) and HbA1c (6.9 +/- 0.6 and 7.1 +/- 0.6%) levels. Possible obstructions and set leakages were infrequently reported in both groups. Similar numbers of patients experienced hypoglycemia (blood glucose <2.5 mmol/l): 14 (74%) insulin aspart patients versus 6 (60%) buffered regular human insulin patients. Patients receiving insulin aspart had fewer hypoglycemic events per patient (2.9) than those patients receiving buffered regular human insulin (6.2). There were no differences between the two insulins in the occurrence of hyperglycemic events (blood glucose >19 mmol/l) or in the number and type of adverse events. CONCLUSIONS: Insulin aspart and buffered regular human insulin were effective and well tolerated and provided similar pump compatibility when used in CSII therapy.

Antipsychotic drugs: importance of dopamine receptors for mechanisms of therapeutic actions and side effects.

Interaction of the antipsychotic drugs with dopamine receptors of the D2, D3, or D4 subclasses is thought to be important for their mechanisms of action. Consideration of carefully defined affinities of the drugs for these three receptors suggests that occupancy of the D4 subclass is not mandatory for achieving antipsychotic effects, but actions at D2 or D3 receptors may be important. A major difference between typical and atypical antipsychotic drugs is in the production of extrapyramidal side effects by the typical drugs. Production of extrapyramidal side effects by typical drugs seems to be due to the use of the drugs at doses where striatal D2 receptor occupancy exceeds approximately 80%. Use of these drugs at doses that do not produce this level of receptor blockade enables them to be used therapeutically without producing these side effects. The antipsychotic drugs have been shown to act as inverse agonists at D2 and D3 dopamine receptors, and this property may be important for the antipsychotic effects of the drugs. It is suggested that the property of inverse agonism leads to a receptor up-regulation upon prolonged treatment, and this alters the properties of dopamine synapses. Several variants of the dopamine receptors exist with different DNA sequences and in some cases different amino acid sequences. These variants may have different properties that alter the effects of dopamine and the antipsychotic drugs. The determination of such variants in patients may help in the prediction of drug responsiveness.

Site-directed mutations in the third intracellular loop of the serotonin 5-HT(1A) receptor alter G protein coupling from G(i) to G(s) in a ligand-dependent manner.

The effect of mutations (V344E and T343A/V344E) in the third intracellular loop of the serotonin 5-HT(1A) receptor expressed transiently in human embryonic kidney 293 cells have been examined in terms of receptor/G protein interaction and signaling. Serotonin, (R)-8-hydroxy-2-dipropylaminotetralin [(R)-8-OH-DPAT], and buspirone inhibited cyclic AMP production in cells expressing native and mutant 5-HT(1A) receptors. Serotonin, however, produced inverse bell-shaped cyclic AMP concentration-response curves at native and mutant 5-HT(1A) receptors, indicating coupling not only to G(i)/G(o), but also to G(s). (R)-8-OH-DPAT, however, induced stimulation of cyclic AMP production only after inactivation of G(i)/G(o) proteins by pertussis toxin and only at the mutant receptors. The partial agonist buspirone was unable to induce coupling to G(s) at any of the receptors, even after pertussis toxin treatment. The basal activities of native and mutant 5-HT(1A) receptors in suppressing cyclic AMP levels were not found to be significantly different. The receptor binding characteristics of the native and mutant receptors were investigated using the novel 5-HT(1A) receptor antagonist [(3)H]NAD-299. For other receptors, analogous mutations have produced constitutive activation. This does not occur for the 5-HT(1A) receptor, and for this receptor the mutations seem to alter receptor/G protein coupling, allowing ligand-dependent coupling of receptor to G(s) in addition to G(i)/G(o) proteins.

Regulation of human D(1), d(2(long)), d(2(short)), D(3) and D(4) dopamine receptors by amiloride and amiloride analogues.

1. The modulatory effects of the allosteric effectors methylisobutylamiloride (MIA), benzamil and amiloride have been examined at human D(1), D(2), D(3) and D(4) dopamine receptors. The subtype selectivity and the mechanism of action of this allosteric regulation was examined. 2. In radioligand dissociation experiments each modulator accelerated dissociation from all four receptor subtypes indicating allosteric regulation. MIA displayed selectivity for the D(3) subtype for acceleration of radioligand dissociation. 3. In equilibrium binding (pseudo-competition) experiments the three compounds inhibited radioligand binding at the four receptor subtypes. Inhibition curves for D(1), D(2(short)), D(2(long)) and D(3) receptors were described by Hill coefficients exceeding unity and data were fitted best by a model that assumes binding of modulator to both the primary and allosteric binding sites of the receptor (the allosteric/competitive model). 4. At the D(4) subtype, Hill coefficients of unity described the binding data for amiloride and benzamil, consistent with competitive inhibition. The Hill coefficient for MIA at the D(4) subtype was less than unity and data could be fitted well by the allosteric/competitive model, but it was not possible to define unambiguously the modulatory mechanism. For this effect a better definition of the mechanism could be obtained by simultaneous analysis of data obtained in the presence of a range of concentrations of a purely competitive ligand. 5. MIA reduced the potency with which dopamine stimulated [(35)S]-GTPgammaS binding at the D(2) receptor. The effects of MIA could be described by the allosteric/competitive model with effects of MIA to inhibit the binding of dopamine but not its ability to induce a response.

Pharmacokinetics of repaglinide in subjects with renal impairment.

OBJECTIVE: To evaluate the effect of renal impairment and renal failure on the pharmacokinetics and safety of repaglinide. METHODS: We conducted a phase I, multicenter, parallel-group, pharmacokinetic comparison trial with single and multiple doses of repaglinide in subjects with various degrees of renal impairment. Subjects with normal renal function (n = 6) and subjects with renal impairment (mild to moderate, n = 6; severe, n = 6) received treatment with 2 mg repaglinide for 7 days. Subjects in the hemodialysis group (n = 6) received two single doses of 2 mg repaglinide separated by a 7- to 14-day washout period. All subjects had repaglinide serum pharmacokinetic profiles measured for the first and last doses administered. Serum steady-state levels, urine levels, and dialysate levels were also measured. RESULTS: Pharmacokinetic parameters did not show significant changes after single or multiple doses of repaglinide, although the elimination rate constant in the group with severe renal impairment decreased after 1 week of treatment. Subjects with severe impairment had significantly higher area under the curve values after single and multiple doses of repaglinide than subjects with normal renal function. No significant differences in values for maximum serum concentration or time to reach maximum concentration were detected between subjects with renal impairment and those with normal renal function. Hemodialysis did not significantly affect repaglinide clearance. CONCLUSIONS: Repaglinide was safe and well tolerated in subjects with varying degrees of renal impairment. Although adjustment of starting doses of repaglinide is not necessary for renal impairment or renal failure, severe impairment may require more care when upward adjustments of dosage are made.